CLINICAL RESEARCH www.jasn.org Urine Metabolite Profiles Predictive of Human Kidney Allograft Status † ‡ | Karsten Suhre,* Joseph E. Schwartz, § Vijay K. Sharma,§ Qiuying Chen, John R. Lee,§ Thangamani Muthukumar,§ Darshana M. Dadhania,§ Ruchuang Ding,§ David N. Ikle,¶ † †† Nancy D. Bridges,** Nikki M. Williams,** Gabi Kastenmüller, Edward D. Karoly, †† ‡‡ || Robert P. Mohney, Michael Abecassis, John Friedewald,§§ Stuart J. Knechtle, || | Yolanda T. Becker, Benjamin Samstein,¶¶ Abraham Shaked,*** Steven S. Gross, and Manikkam Suthanthiran§ *Department of Physiology and Biophysics, Weill Cornell Medical College in Qatar, Doha, Qatar; †Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany; ‡Department of Psychiatry, Stony Brook University, Stony Brook, New York; §Division of Nephrology and Hypertension, Departments of Medicine and Transplantation Medicine, New York Presbyterian Hospital—Weill Cornell Medical Center, New York, New York; |Department of Pharmacology, Weill Cornell College of Medicine, New York, New York; ¶Rho Federal Systems, Chapel Hill, North Carolina; **National Institute of Allergy and Infectious Diseases, Bethesda, Maryland; ††Metabolon, Inc., Durham, North Carolina; ‡‡Division of Surgery-Organ Transplantation, Northwestern University Feinberg School of Medicine, Chicago, Illinois; §§Division of Nephrology- Organ Transplantation, Northwestern University Feinberg School of Medicine, Chicago, Illinois; ||Division of Surgery, Department of Surgery, University of Wisconsin Hospitals and Clinics, Madison, Wisconsin; ¶¶Division of Transplantation, Department of Surgery, Columbia University College of Physicians and Surgeons, New York, New York; and ***Department of Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania ABSTRACT Noninvasive diagnosis and prognostication of acute cellular rejection in the kidney allograft may help realize the full benefits of kidney transplantation. To investigate whether urine metabolites predict kidney allograft status, we determined levels of 749 metabolites in 1516 urine samples from 241 kidney graft recipients enrolled in the prospective multicenter Clinical Trials in Organ Transplantation-04 study. A metabolite signature of the ratio of 3-sialyllactose to xanthosine in biopsy specimen-matched urine supernatants best discriminated acute cellular rejection biopsy specimens from specimens without rejection. For clinical application, we developed a high- throughput mass spectrometry-based assay that enabled absolute and rapid quantification of the 3-sialyllactose-to-xanthosine ratio in urine samples. A composite signature of ratios of 3-sialyllactose to xanthosine and quinolinate to X-16397 and our previously reported urinary cell mRNA signature of 18S ribosomal RNA, CD3« mRNA, and interferon-inducible protein-10 mRNA outperformed the metabolite signatures and the mRNA signature. The area under the receiver operating characteristics curve for the composite metabolite– mRNA signature was 0.93, and the signature was diagnostic of acute cellular rejection with a specificity of 84% and a sensitivity of 90%. The composite signature, developed using solely biopsy specimen-matched urine samples, predicted future acute cellular rejection when applied to pristine samples taken days to weeks before biopsy. We conclude that metabolite profiling of urine offers a noninvasive means of diagnosing and prognos- ticating acute cellular rejection in the human kidney allograft, and that the combined metabolite and mRNA signature is diagnostic and prognostic of acute cellular rejection with very high accuracy. J Am Soc Nephrol 27: 626–636, 2016. doi: 10.1681/ASN.2015010107 Received January 29, 2015. Accepted April 15, 2015. Medical College, Division of Nephrology and Hypertension, Department of Medicine, 525 E. 68th Street, Box 3, New York, NY Published online ahead of print. Publication date available at 10065. Email: [email protected] www.jasn.org. Correspondence: Dr. Manikkam Suthanthiran, Weill Cornell Copyright © 2016 by the American Society of Nephrology 626 ISSN : 1046-6673/2702-626 J Am Soc Nephrol 27: 626–636, 2016 www.jasn.org CLINICAL RESEARCH Kidney transplantation is the preferred treatment for patients patients for metabolomics (Figure 1) to include: (1) all 298 with end stage renal disease, but acute rejection, a frequent and urine samples matched to 298 kidney allograft biopsies (urine serious post-transplant complication, undermines realization samples collected from 3 days before to 1 day after the biopsy); of the full benefits of this intervention. The invasive allograft (2) all 808 sequential urine samples preceding a biopsy diag- biopsy performed to diagnose acute rejection has become safer nosis; and (3) all 412 urine samples from clinically stable pa- over the years, but bleeding and graft loss still occur following a tients who provided .10 sequential samples in the first 400 biopsy. Sampling errors and inter-observer variability in days of transplantation. We obtained high-quality data for biopsy readings pose challenges and the feasibility and cost 1516 urine samples from 241 kidney allograft recipients after of repeated biopsies needed to capture anti-allograft immunity exclusion of one patient and two samples (see Supplemental are major drawbacks. Development of noninvasive biomarkers Material for details) regarding 749 different metabolites from of acute rejection is therefore a major objective of the field. 65 metabolic pathways, including 368 metabolites of un- The multicenter, NIH-sponsored Clinical Trials in Organ known identity (Supplemental Table 1). Transplantation-04 (CTOT-04) investigated whether mRNA levels in urinary cells collected at the time of biopsy are Urine Metabolites and ACR diagnostic of acute rejection and whether mRNA profiles of Metabolite data from 50 urine samples matched to 50 ACR sequential urine specimens obtained at clinically stable time biopsies from 36 patients and 198 urine samples matched to points predict the future development of acute rejection.1 Data 198 No Rejection biopsies from 149 patients were analyzed to from the CTOT-04 study demonstrated that a 3-gene signa- determine whether metabolite profiles distinguish ACR bi- ture of 18S rRNA and 18S-normalized CD3« mRNA and IP-10 opsies from No Rejection biopsies. mRNA (mRNA signature) in urinary cells discriminated acute Supplemental Table 2 lists the characteristics of transplant cellular rejection (ACR) biopsies from biopsies without fea- recipients, such as age, gender, ethnicity, race, and BMI. Among tures of rejection (No Rejection biopsies). Furthermore, there the 50 ACR biopsies graded using the Banff schema, 23 were was a sharp and significant rise in the diagnostic signature graded as ACR grade IA, 11 as ACR grade IB, 12 as ACR grade score during the weeks prior to an ACR biopsy.1 However, IIA, 3 as ACR grade IIB and 1 as ACR grade III. The 198 biopsies despite the progress toward noninvasive characterization of classified as No Rejection biopsies did not show histologic kidney allograft status by mRNA profiling of urine from kid- features of ACR, AMR, borderline, bacterial infection/ ney graft recipients,1,2 there remains huge potential for further pyelonephritis, cytomegalovirus, polyomavirus type BK /polyoma progress by probing the small molecule composition of these nephropathy or post-transplant lymphoproliferative disease. urines for ascertaining kidney allograft status.3 However, several of the No Rejection biopsies displayed histo- Metabolomics aims to measure all relevant small molecules logic changes consistent with ATN (n=79), tubular atrophy (metabolites),3–8 and nontargeted metabolite profiling allows (n=75), interstitial fibrosis (n=67), glomerulosclerosis (n=30), for the relative quantification of hundreds of metabolites in vascular narrowing (n=20), calcineurin inhibitor toxicity small volumes of biologic specimens such as urine. As both in- (n=18), and/or recurrent disease (n=2). Also, several of the bi- termediate and end point markers of diverse biologic processes opsies showed more than one histologic abnormality such as the in the human body, observations of altered metabolite concen- presence of both interstitial fibrosis and tubular atrophy. trations provide access to functionally relevant read-outs of per- Kidney allograft function, measured at the time of biopsy, turbed disease-associated pathways in human metabolism.9–12 showed that the graft function was significantly inferior in the In this investigation, we used cell-free urine supernatants ACR biopsy group compared with the No Rejection biopsy collected from the kidney transplant recipients enrolled in the group. As summarized in Supplemental Table 2, the mean (6 CTOT-04 study to conduct a large-scale study of the urine SD) serum creatinine level at the time of kidney allograft biopsy metabolome to investigate whether urine metabolite profiles in the ACR biopsy group was 3.762.9 mg/dl (number of mea- are diagnostic and prognostic of ACR. Wefurther examined the surements = 37) and was 2.562.3 mg/dl (number of measure- diagnostic and prognostic performance of a combination of ments = 146) in the No Rejection biopsy group (Mann–Whitney metabolites and the previously identified urinary cell mRNA P,0.001), and the mean (6SD) estimated glomerular filtration signature. Finally, we developed a targeted high-throughput rate (eGFR) in the ACR biopsy group was 30617 ml/min per metabolomics assay for